Auxin was the first plant hormone ever identified, and it has been implicated in almost every aspect of plant growth and development. Auxin has been studied for over a century, but the molecular mechanisms that promote its biological activities remain poorly understood. It is not even clear how auxin is produced by plants, let alone how that process is regulated by developmental and environmental signals. This lack of knowledge of auxin biosynthesis further clouds our understanding of auxin-mediated signal transduction. The major objective of the work proposed herein is to isolate and characterize previously unidentified components of auxin biosynthesis and signaling. We have characterized a dominant auxin overproduction mutant, yucca, and this provides us with a unique opportunity to design new strategies to examine auxin-related processes. YUCCA is a member of the flavin-containing monooxygenase (FMO) superfamily, and it catalyzes a rate-limiting step in auxin biosynthesis. Additional components of auxin biosynthesis and signaling can be isolated from genetic screens for yucca suppressors and yucca-like mutants. Biochemical approaches can be utilized to identify YUCCA associated proteins. The first specific aim of this proposal is to characterize yucca suppressors. Second, we propose to further define the roles of the YUCCA gene family by using reverse genetics. Third, we will use biochemical approaches to examine YUCCA associated proteins. Fourth, we will further characterize yucca-like mutants that we have already identified. Fifth and finally, we propose to use chemical genetics in our characterization of sir1, a mutant insensitive to sirtinol that shows constitutive activation of known auxin-inducible genes. These studies are multi-disciplinary in nature and should yield significant new insights into the mechanisms of auxin regulated processes and these processes are of paramount importance to plant biology. A clear understanding of auxin's role in plant growth and development will ultimately have significant agricultural impact. Finally, the proposed study will augment our understanding of complex signaling mechanisms in other eukaryotes, particularly in the area of tryptophan homeostasis, which is essential to most organisms, including humans. [unreadable] [unreadable]